Lc-ms configuration for purification and detection of analytes having a broad range of hydrophobicites

ABSTRACT

Systems, apparatuses, kits, and methods for purification and analysis of analytes having a broad range of hydrophobicities by liquid chromatography-mass spectrometry (LC-MS). Using one set of liquid chromatography columns, one set of mobile phase buffers, and, optionally, a single ionization method (e.g., electrospray ionization), a wide range of analytes can be purified and analyzed on a liquid chromatography-mass spectrometry (LC-MS) system. LC-MS purification and analysis of analytes having a broad range of partition coefficients is accomplished by selecting LC run parameters and MS system parameters that are particular to different classes of analytes without having to make column or buffer changes or any other hardware configuration changes to the LC-MS system. The methods, systems, and kits described herein provide for substantially increased speed/throughput and ease of use for a wide range analytes with essentially no compromise in specificity for individual analytes relative to previously described methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of and priority to U.S. Prov. Pat.App. Ser. No. 61/408,266 entitled “LC-MS CONFIGURATION FOR PURIFICATIONAND DETECTION OF ANALYTES HAVING A BROAD RANGE OF HYDROPHOBICITIES”filed 29 Oct. 2010 with inventors Joseph L. Herman, Robert DeWitte, andDayana Argoti, the entirety of which is incorporated herein byreference. This Application also references U.S. Prov. Pat. App. Ser.No. 61/408,385 entitled “LC-MS SEPARATION AND DETECTION OF VITAMIN DMETABOLITES” filed 29 Oct. 2010 with inventors Joseph L. Herman andDayana Argoti, the entirety of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION 1. The Field of the Invention

The invention relates to methods, systems, apparatuses, and kits forLC-MS separation and detection of analytes

2. The Relevant Technology

Liquid chromatography-mass spectrometry (LC-MS) is a powerful analytedetection and measurement technique that is quickly becoming thepreferred method of detecting small molecule analytes for diagnosticpurposes. However, the instrumentation required for LC-MS is technicallycomplex and, as such, is typically not well suited to the averagehospital clinical lab or medical lab technologist. By and large, theselabs have not adopted LC-MS diagnostics and, instead, generally usealternative diagnostic techniques, including automated immunoassay, orsend the samples out to a reference laboratory for analysis. Moreover,even reference laboratories or other types of testing laboratoriesincluding, without limitation, those for testing industrial orenvironmental samples would make broader use of LC-MS if the procedurewas simplified and substantially automated.

Current LC-MS methods require careful selection of the appropriateliquid chromatography column and mobile phases for each differentanalyte of interest, as well as careful calibration of the massspectrometer to isolate and identify the analyte of interest. In orderto analyze a second different analyte on the same instrument, one ormore of the column, the mobile phases, or the mass spectrometer settingsgenerally must be changed and optimized by the LC-MS technologist.Because of the time and technical complexity of such changes, randomaccess analysis of individual samples is costly and inefficient. Soinstead, samples flagged for analysis for the same or similar analytesare generally grouped into large batches and run together. While thisarrangement may reduce the number of changes to the LC-MS system set-upfrom run-to-run, it significantly increases the time to result for eachsample. The high complexity of the LC-MS setup and process calls forhaving an expert LC-MS technologist on hand all the time to makeadjustments, manual changes, and hardware re-configurations to thesystem.

Since hospitals are typically not equipped or staffed to perform suchsophisticated analytical chemistry experiments, LC-MS systems aregenerally not available at hospitals today. Instead, samples flagged forLC-MS analysis are sent out to a few central reference labs wheresamples are batched according to the ordered assay type(s). Thispractice is time-consuming and expensive. In addition, this situationmakes it difficult for multiple analyses to be performed on the samesample. As a result, samples may be held for several hours or even daysbefore their batch is analyzed and samples containing multiple analytesof interest may have to be aliquoted separately or placed back into thequeue batch each time a different analyte is to be assayed. If thehospital is not near a major reference lab having LC-MS equipment, onemust transport the sample to and from the lab, creating a further delayof possibly days. For time sensitive analyses (e.g., for emergencydepartment patients or for samples containing unstable analytes), suchdelays are unacceptable. For more routine tests, such delays and addedexpense render many powerful LC-MS diagnostic tests simply unavailabletoday to many hospitals and diagnostic laboratories.

Efforts have been made to simplify LC-MS analysis by reducing thenumbers of different columns and mobile phase buffers needed to purifyand analyze a wide range of compounds having different characteristics.For example, Herman reported (Rapid Commun. Mass Spectrom. 16: 421-426,2002) an LC-MS method to analyze a library of drug compounds for thepurpose of drug-screening assays, not for diagnostic purposes, using oneset of columns, one set of mobile phase buffers, and one set of liquidchromatography conditions (e.g., flow rate, isocratic elution, etc). Themethod of Herman is applicable to compounds having hydrophobicitiesspanning about 4 log partition coefficient (logP) units whereindetection within established clinical reference ranges and/or conformityto clinical standards are not required. However, the method of Herman isnot applicable to extremely hydrophilic compounds (i.e., logP less thanabout 1) or to extremely hydrophobic compounds (i.e., logP greater thanabout 5).

BRIEF SUMMARY

The present invention relates to methods, systems, apparatuses, and kitsfor purification and detection of analytes having a broad range ofhydrophobicities by liquid chromatography-mass spectrometry (LC-MS).Using one set of liquid chromatography columns, one set of mobile phasebuffers, and, optionally, a single ionization method (e.g., electrosprayionization), a wide range of analytes can be purified and analyzed on aliquid chromatography-mass spectrometry (LC-MS) system. LC-MSpurification and analysis of analytes having a broad range ofhydrophobicities is accomplished by selecting LC run parameters (e.g.,flow rate, ratios of one buffer to another, temperature, and the like)and MS system parameters (e.g., ionization voltage, desolvationtemperature, and the like) that are particular to different analyteswithout having to make column or buffer changes or any other hardwareconfiguration changes to the LC-MS system. The methods, systems,apparatuses, and kits described herein provide for substantially reducedtime to results when multiple samples are analyzed in no particularorder. The methods, systems, apparatuses, and kits described herein alsoprovide order and ease of use for a wide range analytes with essentiallyno compromise in specificity for individual analytes relative topreviously described methods.

In one embodiment, a method for detecting and/or quantifying at leasttwo analytes with widely different hydrophobicities using liquidchromatography-mass spectrometry (LC-MS) is disclosed. The methodincludes (1) providing two or more analytes selected from a first groupand a second group, wherein the log partition coefficients (logP) of thetwo or more analytes selected from the first group and the second groupare separated by at least about 4.5 logP units, (2) purifying at leastone analyte from each of the first and second groups using a singleanalytical liquid chromatography column of a liquid chromatographysystem, one aqueous mobile phase buffer solution of the liquidchromatography system, and one substantially non-aqueous mobile phasebuffer solution of the liquid chromatography system, and (3) analyzingthe analytes from the first and second groups using a mass spectrometer.In one embodiment, the mass spectrometer may be in fluid communicationwith the liquid chromatography system. In another embodiment, the massspectrometer may be “offline” relative to the liquid chromatographysystem.

The analytes can be contained in the same sample or they can becontained in different samples. Likewise, the analytes can be purifiedand analyzed in a single LC-MS run or they can be analyzed at differenttimes.

No changes to the LC-MS system hardware are necessary in order toserially purify and analyze analytes separated by about 4.5 or more logPunits. That is, it is not necessary to change hardware parameters likecolumns and mobile phase buffers in order to purify and analyzedifferent analytes. Instead, LC-MS system parameters such as, but notlimited to, mobile phase buffer flow rate, a ratio of an aqueous mobilephase buffer solution to a non-aqueous mobile phase buffer solution, agradient varying ratios of the aqueous and non-aqueous mobile phasebuffer solutions, ionization voltage, desolvation temperature, electrodevoltage, collision gas temperature, collision gas pressure, collisionenergy, and combinations thereof can be changed depending on theanalyte.

In another embodiment, a method for detecting and/or quantifying two ormore analytes with widely different hydrophobicities using LC-MS isdisclosed. The method includes (1) providing a single analytical liquidchromatography column of a liquid chromatography-mass spectrometry(LC-MS) system, one aqueous mobile phase buffer solution of the LC-MSsystem, and one substantially non-aqueous mobile phase buffer solutionof the LC-MS, wherein the LC-MS system is configured for purifying andanalyzing a plurality of analytes having a log partition coefficients(logP) spanning a range of about −1.2 to about 6 by varying at least oneLC-MS system parameter, (2) selecting at least two analytes that spanthe log partition coefficient range from about −1.2 to about 6; (3)selecting at least one analysis protocol based on the selected analytes,wherein selecting the at least one analysis protocol includes varying atleast one LC-MS system parameter, and (4) purifying and analyzing theselected analytes using the LC-MS and the selected analysis protocols,such that a diagnostic quality standard is satisfied for each of the twoanalytes.

In yet another embodiment, a method is disclosed. The method includes(1) providing a first analyte and a second analyte selected from thegroup consisting of vitamin D, steroid hormones, protein hormones,proteins, peptides, immunosuppressants, chemotherapeutics, tricyclicantidepressants, azole antifungals, anti-epileptics, anti-retrovirals,opiates and/or opioids, drugs of abuse, barbiturates, benzodiazepines,or a metabolite thereof, wherein the log partition coefficients (logP)of the first and second analytes are separated at least about 4.5 logPunits. The method further includes (2) purifying the first analyte andthe second analyte using a single analytical liquid chromatographycolumn of a liquid chromatography system, one aqueous mobile phasebuffer solution of the liquid chromatography system, and onesubstantially non-aqueous mobile phase buffer solution of the liquidchromatography system, and (3) analyzing the first analyte and thesecond analyte using a mass spectrometer that is in fluid communicationwith the liquid chromatography system.

In still yet another embodiment, a method for detecting and/orquantifying three or more analytes with widely differenthydrophobicities using LC-MS is disclosed. The method includes (1)providing a first analyte, a second analyte, and a third analyte,wherein the first analyte has a log partition coefficient (logP) in arange of about −1.2 to about 0, the second analyte has a logP in a rangefrom about 0 to about 5, and the third analyte has a logP greater thanabout 5 and less than or equal to about 6. In total, this represents thealmost the entire practical range of logPs with the first analyte beingvery hydrophilic and the third analyte being very hydrophobic and thesecond analyte being essentially anywhere in between.

The method further includes (2) providing a single analytical liquidchromatography column of a liquid chromatography-mass spectrometry(LC-MS) system, one aqueous mobile phase buffer solution of the LC-MSsystem, and one substantially non-aqueous mobile phase buffer solutionof the LC-MS, and a mass spectrometer, and (3) purifying and analyzingthe first, second, and third analytes on the LC-MS system. One willappreciate that the first, second, and third analytes can be analyzed inessentially any order.

In still yet another embodiment, a system for purifying and analyzinganalytes of interest by LC-MS is disclosed. The system includes a singleanalytical liquid chromatography column of a liquid chromatographysystem, one aqueous mobile phase buffer solution of the liquidchromatography system, and one substantially non-aqueous mobile phasebuffer solution of the liquid chromatography system, and a massspectrometer capable of ionizing, fragmenting, and detecting one or moreparent ions or product ions specific to each analyte purified and elutedfrom the liquid chromatography system.

The liquid chromatography system is capable of effecting purificationand elution of each of vitamin D, steroid hormones, protein hormones,proteins, peptides, immunosuppressants, chemotherapeutics, tricyclicantidepressants, azole antifungals, anti-epileptics, anti-retrovirals,opiates and/or opioids, drugs of abuse, barbiturates, benzodiazepines,metabolites thereof, and combinations thereof with the single analyticalliquid chromatography column, the one aqueous mobile phase buffersolution, and the one substantially non-aqueous mobile phase buffersolution.

In still yet another embodiment, a kit for purifying and analyzinganalytes of interest by LC-MS is disclosed. The kit includes at leastone analytical liquid chromatography column, reagents for purifying aplurality of analytes using a liquid chromatography system, wherein thereagents comprise at least one aqueous mobile phase buffer solution andat least one substantially non-aqueous mobile phase buffer solution, anda protocol for analyzing the plurality of analytes using a massspectrometer. The protocol includes instructions for purifying andanalyzing analytes having partition coefficients (logP) ranging fromabout −1.2 to about 6 using a single analytical liquid chromatographycolumn, a single aqueous mobile phase buffer, and a single substantiallynon-aqueous mobile phase buffer. In one embodiment, the protocol furtherincludes instructions for purifying and analyzing the analytes havinglogPs ranging from about −1.2 to about 6 such that a diagnostic qualitystandard is satisfied for each analyte in the logP range from about −1.2to about 6.

In still yet another embodiment, an apparatus for purifying andanalyzing analytes of interest having a broad range of hydrophobicitiesis disclosed. The apparatus includes a single analytical liquidchromatography column of a liquid chromatography system, one aqueousmobile phase buffer solution of the liquid chromatography system, andone substantially non-aqueous mobile phase buffer solution of the liquidchromatography system, wherein the liquid chromatography system iscapable of effecting purification and elution of analytes spanning a logpartition coefficient (logP) range of about −1.2 to about 6 using thesingle analytical liquid chromatography column, the one aqueous mobilephase buffer solution, and the one substantially non-aqueous mobilephase buffer solution.

The apparatus further includes a mass spectrometer capable of ionizing,fragmenting, and detecting one or more parent ions or product ionsspecific to each analyte purified and eluted from the liquidchromatography system, a sample handling device configured to manage aplurality of samples, and a control system linked to each of the liquidchromatography system and the mass spectrometer and configured tocontrol or vary at least one LC-MS system parameter (i.e., a softwarecontrolled parameter and not a hardware parameter).

These and other objects and features of the present invention willbecome more fully apparent from the following description and appendedclaims, or may be learned by the practice of the invention as set forthhereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent invention, a more particular description of the invention willbe rendered by reference to specific embodiments thereof which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only illustrated embodiments of the invention and aretherefore not to be considered limiting of its scope. The invention willbe described and explained with additional specificity and detailthrough the use of the accompanying drawings in which:

FIG. 1 is a block diagram schematically illustrating a system forpurifying and analyzing analytes having a broad range of partitioncoefficients;

FIG. 2 is a flow diagram illustrating a method for purifying andanalyzing analytes having a broad range of partition coefficients byliquid chromatography-mass spectrometry (LC-MS);

FIG. 3 is a flow diagram illustrating a method for purifying andanalyzing analytes having a broad range of partition coefficients byliquid chromatography-mass spectrometry (LC-MS);

FIG. 4A depicts an ESI full scan spectrum of the molecular ion regionfor 25-OH D₂ using ammonium formate;

FIG. 4B depicts an ESI full scan spectrum of the molecular ion regionfor 25-OH D₂ using formic acid;

FIG. 5A depicts the selected reaction monitoring (“SRM”) signal from25-OH D₂ using ESI to produce the [M+H]+ molecular ion;

FIG. 5B depicts the SRM signal from 25-OH D₂ using APCI to produce the[M+H—H₂O]+ water loss ion;

FIG. 6 depicts MRM chromatograms from an injection of a solutioncontaining morphine, norcodeine, hydromorphone, and norhydrocodone;

FIG. 7 depicts MRM chromatograms from an injection of a solutioncontaining of codeine, oxycodone, 6-monoacetylmorphine (6-MAM), andhydrocodone;

FIG. 8 depicts MRM chromatograms from an injection a solution containingoxymorphone and noroxycodone;

FIG. 9 depicts MRM chromatograms from an injection of a solutioncontaining tapentadol, norfentanyl, metoprolol, fentanyl,benzoylecgonline, cocaine, and phencyclidine (PCP);

FIG. 10 depicts MRM chromatograms from an injection of a solutioncontaining dihydrocodeine, d⁹-THC-COOH, d⁹-THC, tramadol, and methadone;

FIG. 11 depicts MRM chromatograms from an injection of a solutioncontaining the immunosuppressant drugs tacrolimus, everolimus,sirolimus, and cyclosporine A;

FIG. 12 depicts MRM chromatograms from an injection of a solutioncontaining the steroids cortisone, progesterone, prednisone,androstenedione, cortisol (hydrocortisone), hydroxyprogesterone, andtestosterone;

FIG. 13 depicts MRM chromatograms from an injection of a solutioncontaining 25-hydroxy vitamin D₂ and D₃;

FIG. 14 depicts MRM chromatograms from an injection of a solutioncontaining the amphetamines amphetamine, MDMA, and methamphetamine;

FIG. 15 depicts MRM Chromatograms from an injection of a solutioncontaining the chemotherapeutic drugs methotrexate, docetaxcel, andbusulfin;

FIG. 16 depicts MRM chromatograms from an injection of anantibody-stripped serum solution containing norfentanyl, tapentadol,tramadol, cocaine, fentanyl, PCP, THC-COOH, and methadone;

FIG. 17 depicts MRM chromatograms from an injection of anantibody-stripped serum solution containing norcodeine, codeine,dihydrocodeine, amphetamine, benoylecgonine, MDA, methamphetamine, MDMA,and MDEA;

FIG. 18 depicts MRM chromatograms from an injection of anantibody-stripped serum solution containing morphine, oxymorphone,hydromophone, noroxycodone, and norhydrocodone;

FIG. 19 depicts MRM chromatograms from an injection of anantibody-stripped serum solution containing 6-MAM, oxycodone, andhydrocodone;

FIG. 20 depicts MRM chromatograms from an injection of anantibody-stripped serum solution containing tacrolimus, everolimus,sirolimus, and cyclosporin A;

FIG. 21 depicts standard curves generated from a whole blood solutioncontaining the immunosuppressant drugs tacrolimus, everolimus, andsirolimus;

FIG. 22 depicts standard curves generated from a plasma solutioncontaining 25-hydroxy Vitamin D₂ and D₃;

FIG. 23 depicts a standard curve for docetaxcel in plasma;

FIG. 24 depicts a standard curve for busulfin in plasma

FIG. 25 depicts a standard curve for cyclosporin A in antibody-strippedserum; and

FIG. 26 depicts a standard curve for testosterone in antibody-strippedserum.

DETAILED DESCRIPTION I. Introduction and Definitions

The present invention relates to methods, systems, and kits forpurification and detection of analytes having a broad range ofhydrophobicities by liquid chromatography-mass spectrometry (LC-MS).Using a single liquid chromatography column (e.g., a single analyticalliquid chromatography column) or a single set of liquid chromatographycolumns (e.g., a single sample clean-up liquid chromatography column influid communication with and upstream of the single analytical liquidchromatography column), one set of mobile phase buffers, and,optionally, a single ionization method (e.g., electrospray ionization(“ESI”), atmospheric pressure chemical ionization (“APCI”) and otherionization methods as known in the art), a wide range of analytes can bepurified and analyzed on a liquid chromatography-mass spectrometry(LC-MS) system. LC-MS purification and analysis of analytes having abroad range of hydrophobicities is accomplished by selecting LC runparameters (e.g., flow rate, ratios of one buffer to another,temperature, and the like) and MS system parameters (e.g., ionizationvoltage, desolvation temperature, and the like) that are particular todifferent analytes without having to make column or buffer changes orany other hardware configuration changes to the LC-MS system. Due to thefact that columns, buffers, and the like do not need to be changedbetween runs with different analyte types, the methods, systems, andkits described herein provide for substantially increased speed and easeof use for a wide range analytes with essentially no compromise inspecificity for individual analytes relative to previously describedmethods.

As used herein, the terms “partition coefficient” and “logP” refer theratio of concentrations of a compound between two immiscible solvents(e.g., octanol and water) at equilibrium. Hence, to a firstapproximation, LogP is a measure of how hydrophilic (“water loving”) orhydrophobic (“water fearing”) a chemical substance is. It should benoted that logP is an exponential function, thus, in general, a changeof one unit represents a factor of 10 difference in hydrophobicity.

Purifying and analyzing analytes having a broad range ofhydrophobicities, which are estimated by logP measurements, posesparticular challenges with current LC-MS technology. This is due inlarge part to challenges associated with achieving chromatographicseparation of such broad ranging compounds. Typically, LC-MS systemsseparate compounds for analysis in the mass spectrometer using ahigh-performance liquid chromatography (HPLC) system, which separatescompounds based on their hydrophobicity by taking advantage of changesin the partitioning between the stationary and mobile phases. Largedifferences in hydrophobicity tend to result in vastly differentretention characteristics on HPLC.

Generally, a result of this is that changes in the mobile phase (i.e.,liquid buffer solutions), stationary phase (column selection), or bothare needed to achieve the desired chromatographic separation whenworking with compounds from different chemical classes (e.g., differentand broad ranging logPs). That is, liquid chromatography hardware (e.g.,columns), consumables (e.g., mobile phase buffers), MS set up (e.g.,ionization type), and systems parameters (e.g., flow rate, gradients,etc.) are generally optimized to each different analyte type or class ofanalyte, where classes are defined by a fairly narrow range of logPswithin each given class.

As a consequence, samples to be analyzed are currently grouped togetheraccording to the characteristics of the analyte(s) of interest andsimilar samples are run together in batches on an instrument equippedwith a particular set of columns, buffers, and run parameters that areunique to the analyte of interest. In order to run samples containing asecond, different analyte of interest, one or more of the column, themobile phase buffers, or the run parameters generally must generally bechanged and optimized by the LC-MS operator. While this arrangement mayreduce the number of changes to the LC-MS system set-up from run-to-run,it significantly increases the time to result for each sample. Inaddition, this situation makes it difficult for multiple analyses to beperformed on the same sample. As a result, samples may have to waitseveral hours or even days before their batch is analyzed and samplescontaining multiple analytes of interest have to be divided intoseparate aliquots or placed back into the queue each time a differentanalyte is to be assayed. For time sensitive analyses (e.g., foremergency department patients or for samples containing unstableanalytes), such delays are unacceptable.

The present invention seeks to simplify LC-MS purification and analysisof analytes from multiple and broad ranging chemical classes (e.g., abroad range of log partition coefficients) by presenting methods,systems, apparatuses, and kits that are configured for assaying (i.e.,purifying and analyzing) compounds having a broad range ofhydrophobicities using a single set of liquid chromatography columns(e.g., a clean-up column and an analytical column) and a single set ofmobile phase buffers (e.g., an aqueous buffer and a non-aqueous buffer).And instead of changing columns and buffers when switching from onechemical class to another, LC-MS purification and analysis isaccomplished by selecting LC run parameters (e.g., flow rate, ratios ofone buffer to another, temperature, and the like) and MS systemparameters (e.g., ionization voltage, desolvation temperature, and thelike) that are particular to each different analytes or classes ofanalytes.

The difficulty in developing a unified LC-MS method that can be used topurify and assay compounds having a broad range of logPs by LC-MS can beillustrated by reference to Table 1 (below).

TABLE 1 logP Endocrinology Vitamin D 25OH vitamin D₂ 5.69 25OH vitaminD₃ 5.61 Steroids Testosterone 4.44 Cortisol (hydrocortisone) 1.26Cortisone 1.58 Progesterone 3.58 Hydroxyprogesterone 3.52 Prednisone2.07 Androstenedione 2.93 Therapeutic Drug Monitoring ImmunosuppressantsTacrolimus 3.3 Everolimus 5.01 Sirolimus 4.3 Cyclosporin A 4.12Chemotherapeutics Methotrexate 0.94 Busulfan −1.15 5-Fluorouracil −0.9Docetaxel 2.4 Pain management & Drugs of Abuse NIDA 5 Phencyclidine 4.14Benzoylecgonine 1.64 Cocaine 1.91 Delta9-THC 5.53 11-norDelta-9-THC-COOH4.6 Amphetamines Amphetamine 1.7 Methamphetamine 2.2 MDMA 1.98 MDEA 2.31MDA 1.46 Opiates/Opioids Hydromorphone 1 Norhydrocodone 0.89 Norcodeine1.07 Morphine 1.73 Hydrocodone 1.27 Codeine 1.45 Noroxycodone 0.1Oxymorphone 0.21 Dihydrocodeine 1.63 Oxycodone 0.48 6-MAM 1.81Tapentadol 3.43 Norfentanyl 3.94 Fentanyl 4.59 Tramadol 2.53 Methadone4.55 Metoprolol 2.18The compounds listed in Table 1 can be analyzed by LC-MS for a varietyof clinical and drug monitoring purposes. Under current practice,however, LC-MS analysis relies on using protocols that are particular tothe different classes of analytes. The compounds listed in Table 1 havelogPs ranging from about −1.2 (busulfan) to about 6 (25OH vitamin D₂),which represents a difference in hydrophobicity over slightly more thanabout seven orders of magnitude. This is a very broad range ofhydrophobicities and it is surprising and unexpected that such a broadrange of analytes can be purified and analyzed by LC-MS using one set ofliquid chromatography columns, one set of mobile phase buffers, and,optionally, a single ionization method.

As used herein, the term “purification” does not refer to removing allmaterials from the sample other than the analyte(s) of interest.Instead, in one aspect, purification may refer to a procedure thatenriches the amount of one or more analytes of interest relative to oneor more other components of the sample. In another aspect, purificationcan be used to remove one or more interfering substances, e.g., one ormore substances that would interfere with detection of an analyte ion bymass spectrometry.

As used herein, “sample” refers to any fluid or liquid sample, includingextractions of solid materials or swabs from environmental surfaces and“biological sample” refers to any sample from a biological source suchas, but not limited to, hair, bodily tissue (e.g., skin or tissuebiopsy), blood, plasma, deproteinated plasma, serum, deproteinatedserum, sputum, bile, saliva, urine, feces, tears, perspiration, swabsfrom body sites, suspensions of microorganisms, and the like.

As used herein, “kit” refers to two or more components comprisingreagents, devices, calibrators, controls, standards, or any combinationthereof, for performance of a common method, regardless of whether thetwo or more components are provided within a single package or multiplepackages.

As used herein, “chromatography” refers to a process in which a chemicalmixture carried by a liquid, gas or supercritical fluid is separatedinto components as a result of differential distribution of the solutesas they flow around or over a stationary phase or chemically interactwith a liquid or solid phase.

As used herein, “liquid chromatography” (LC) means a process ofselective retention of one or more components of a fluid solution as thefluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retention results fromthe distribution of the components of the mixture between one or morestationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). “Liquid chromatography”includes, without limitation, reverse phase liquid chromatography(RPLC), high performance liquid chromatography (HPLC), ultra highperformance liquid chromatography (UHPLC), supercritical fluidchromatography (SFC) and ion chromatography.

As used herein, the term “HPLC” or “high performance liquidchromatography” refers to liquid chromatography in which the degree ofseparation is increased by forcing the mobile phase under pressurethrough a stationary phase, typically a densely packed column.

As used herein, the term “UHPLC” or “ultra high performance liquidchromatography” refers to a liquid chromatography technique similar toHPLC except the operating pressures are higher than HPLC (e.g., about100 MPa vs. about 40 MPa), the columns are typically smaller indiameter, the particles of packing material are generally smaller, andresolution can be greater.

As used herein, “mass spectrometry” (MS) refers to an analyticaltechnique to filter, detect, identify and/or measure compounds by theirmass to charge ratio, of “Dale” (also commonly denoted by the symbol“m/z”). MS technology generally includes (1) ionizing the compounds andpotentially fragmenting the compounds; and (2) detecting the molecularweight of the charged compound and/or fragment ion and calculating amass-to-charge ratio (Dale). The compound may be ionized and detected byany suitable means. A “mass spectrometer” generally includes an ionizerand an ion detector.

The term “ESI” or “electrospray ionization” refers to a technique usedin mass spectrometry to produce ions. It is especially useful inproducing ions from macromolecules because it overcomes the propensityof these molecules to fragment when ionized. In ESI a stream of fluid isejected from a nozzle, cone or other directive device which may or maynot be electrically charged. Molecular ions (e.g., [M+H]+) may be formedin the liquid phase or as a function of the physic-chemical processesoccurring during evaporation of the solvent shell around the analyte orin the gas phase.

The term “ionization” as used herein refers to the process of generatingan analyte ion having a net electrical charge. Negative ions are thosehaving a net negative charge, while positive ions are those having a netpositive charge.

The term “operating in negative ion mode” refers to those massspectrometry methods where negative ions are detected. Similarly,“operating in positive ion mode” refers to those mass spectrometrymethods where positive ions are detected.

The term “desorption” as used herein refers to the removal of an analytefrom a surface and/or the entry of an analyte into a gaseous phase.

The term “about” as used herein in reference to quantitativemeasurements, refers to the indicated value plus or minus 10%.

II. Systems and Apparatuses for LC-MS Purification and Detection ofAnalytes having a Broad Range of Hydrophobicities

Referring now to FIG. 1, a system 100 for LC-MS purification anddetection of analytes from a broad range of chemical classes isschematically illustrated. The system 100 includes a liquidchromatography system 102 capable of effecting purification of analytesfrom a broad range of chemical classes and a mass spectrometer 150capable of ionizing, fragmenting, and detecting one or more precursorions or product ions specific to each analyte of interest.

The liquid chromatography system 102 schematically illustrated in FIG. 1includes a sample 110, reagents 120 for purifying a wide variety ofanalytes of interest from the sample 110 by liquid chromatography, aliquid chromatography column 140 capable of effecting separation ofanalytes having a broad range of hydrophobicities, and a fluid handlingpump 130 in fluid communication with the sample 110, reagents 120, andthe liquid chromatography column 140. The liquid chromatography column140 can include a single liquid chromatography column (e.g., a singleanalytical liquid chromatography column) or a single set of liquidchromatography columns (e.g., a single sample clean-up liquidchromatography column in fluid communication with and upstream of thesingle analytical liquid chromatography column).

In one embodiment, the reagents 120 include a single liquidchromatography buffer. In another embodiment, the reagents 120 mayinclude a single substantially aqueous buffer, a single substantiallynon-aqueous buffer, and at least one wash buffer. In one embodiment, thesingle substantially aqueous buffer and the single substantiallynon-aqueous buffer may include a source of ammonium ions (e.g., ammoniumformate or ammonium acetate). The reagents 120 may also include one ormore reagents for sample preparation, such as a protein precipitationreagent selected for removing proteins that may interfere with assayingan analyte of interest.

As illustrated in FIG. 1, the system 100 further includes an optionalcontrol unit 160 that can be linked various components of the system 100through linkages 170 a-170 d. For example, the control unit 160 can belinked to the sample 110 to control sample application, the reagents 120to control the application of various reagents, the pump 130 to controlfluid handling, flow rates, etc, and to the mass spectrometer 150 tocontrol mass spectrometry parameters. In the illustrated embodiment, thecontrol unit 160 can also serve as a data processing unit to, forexample, process data from the mass spectrometer 150.

In some embodiments, the system 100 is designed to be used by aclinician or a general laboratory technician who is not necessarilyexpert in all aspects of sample preparation, LC-MS operations, and/orLC-MS methods development. As such, the control unit 160 can be designedto encapsulate the LC/MS data system environment by providing a userwith a simplified application interface that can be used to initiate andmonitor essentially all aspects of assaying a sample 110 withoutrequiring the user to interact with the overall hardware and controlsystems of the system 100. The control unit 160 is therefore configuredto provide a degree of separation between the user and the underlyingservices that control devices, data files and algorithms for translatingdata to a user readable form. That is, the control unit 160 eliminatesthe need for the user to be aware of or in control of hardware foranalyzing clinical samples and provides a simplified interface to sendand receive information from the mass spectrometer.

The control unit 160 may be configured to internally monitor each sampleanalysis request and is capable of tracking the analysis request fromstart to finish through the system 100. Once data for a sample 110 hasbeen acquired by the system 100, the control unit 160 may be configuredto automatically start post processing the data based on the type ofassay selected by the user. Moreover, the control unit 160 can beconfigured to automatically select post processing parameters based onthe type of assay selected by the user, further reducing the need forthe user to interact with the system once the assay has been selectedand started for analysis. The control unit 160 can be designed as alayer that fits between an LC/MS system 100 and the user to reduce thecomplexity needed to set up sample assays for acquisition. The controlsystem 160 can also be configured to return only the most relevant datato the user to avoid overwhelming the user with extraneous information.

The control unit 160 can be configured to control the flow ofinformation through the system 100. The following list provides anabbreviated overview of information flow through the system 100 invarious embodiments:

-   -   i. The user submits a sample 110 to the system 100. The system        100 can be set up such that the user then submits an assay        request through the control system 160. Alternatively, the        control system 160 can be linked to a sample detection device        (e.g., a bar code scanner or an RFID scanner) that detects the        presence of the sample and the type of analysis ordered and that        automatically submits an assay request through the control        system 160.    -   ii. The control system 160 translates the assay data and submits        it to the LC-MS system 100. Among other things, this can be used        by the system 100 to set up and initiate the assay parameters        (e.g., flow rate, a ratio of the aqueous mobile phase buffer        solution to the non-aqueous mobile phase buffer solution, a        gradient varying ratios of the aqueous and non-aqueous mobile        phase buffer solutions, ionization voltage, desolvation        temperature, timing and magnitude of voltages applied to various        mass spectrometer electrodes, collision energy, collision gas        temperature, collision gas pressure, etc.).    -   iii. The control system 160 can monitor the status of the sample        collection from the LC/MS system and, once completed, initiate        data processing to generate a result specific for the assay type        selected by the user.    -   iv. When the assay and the data analysis are complete, the        control system 160 can notify the user that the results are        complete for the submitted sample and send the results to the        user. The user can then typically display the results for the        assayed compound either numerically or graphically.

In one embodiment, the system can further include a sample handlingdevice 115 configured to manage a plurality of samples. The samplehandling device 115 can include a carrousel or tray with multiple sampleracks. In turn, each sample rack can include one or more samplecontainer positions capable of holding a sample container, e.g., a tube.

In one embodiment, the system 100 can further include a sample detectiondevice (not pictured) operably coupled to or integrated with the samplehandling device 115. The sample detection device can work with thesample handling device 115 or independently of the sample handlingdevice 115 perform at least one of the following functions:

-   -   i. identify samples entering the system;    -   ii. identify an analyte of interest in the samples entering the        system;    -   iii. select an assay protocol based on the analyte of interest;    -   iv. direct the sample handling device and/or the control system        to initiate analysis of the analyte of interest in the sample;    -   v. direct the control system to select one or more reagents        based upon the assay protocol selected for the analyte of        interest;    -   vi. direct the control system to select a liquid chromatography        mobile phase condition based upon the assay protocol selected        for the analyte of interest and cause the liquid chromatography        system to purify the analyte of interest;    -   vii. direct the control system to select a mass spectrometer        setting based upon the assay protocol selected for the analyte        of interest and cause the mass spectrometer to create mass        spectral data associated with the selected analyte of interest;        or    -   viii. direct the control system to analyze the mass spectral        data associated with the selected analyte of interest to        identify the presence and/or concentration of the analyte of        interest.

Additional discussion of an example of an automated LC-MS system can befound in U.S. Provisional Patent Application Ser. No. 61/408,180entitled “AUTOMATED SYSTEM FOR SAMPLE PREPARATION AND ANALYSIS,” filed29 Oct. 2010 with inventors Robert DeWitte, Juhani Siidorov, VesaNuotio, Raimo Salminen, Jarmo Vehkomäki, Jukka Saukkonen, Bill Östman,Joe Senteno, John Edward Brann III, Joseph L. Herman, and Jeffrey A.Zonderman, the entirety of which is incorporated herein by reference.

Suitable samples 110 include any sample that may contain an analyte ofinterest, such as, but not limited to, biological samples and so-called“neat” samples that contain an analyte or analytes of interest dissolvedin a suitable solvent. For example, samples obtained during themanufacture of an analyte can be analyzed to determine the compositionand yield of the manufacturing process. Samples from environmentalsources include, without limitation, water, toxins, and swabs fromenvironmental surfaces. In certain embodiments, a sample is a biologicalsample; that is, a sample obtained from any biological source, such asan animal, a cell culture, an organ culture, etc. Particularly preferredare samples obtained from a human, such as a blood, plasma,deproteinated plasma, serum, sputum, muscle, urine, saliva, tear,cerebrospinal fluid, swabs from body sites, suspensions ofmicroorganisms or tissue sample. Such samples may be obtained, forexample, from a patient; that is, a living person presenting themselvesin a clinical setting for diagnosis, prognosis, or treatment of adisease or condition.

Samples may be processed or purified to obtain preparations that aresuitable for the desired type of chromatography and/or for analysis bymass spectrometry. Various procedures may be used for this purposedepending on the type sample or the type of chromatography. Examplesinclude filtration, extraction, precipitation, centrifugation, dilution,combinations thereof and the like. Protein precipitation is one examplemethod of preparing a liquid biological sample, such as serum or plasma,for chromatography. In one embodiment, a volume of the liquid sample isadded to a sufficient volume of methanol to cause precipitation of mostof the proteins in the sample while the analyte of interest is left inthe resulting supernatant. The samples can then be centrifuged toseparate the liquid supernatant from the pellet. The resultantsupernatants can then be applied to liquid chromatography and massspectrometry analysis. In some embodiments, the system 100 includes aquality control standard that can be used to track at least one of thehandling, separation, ionization, fragmentation, or detection of theanalyte(s) of interest. For example, hexadeuterated 25-OH D₃ (d₆-25-OHD₃) may be used as a quality control standard or an internal standard inassays for vitamin D and vitamin D metabolites. Persons having skill inthe art can select suitable quality control standards or internalstandards for use with the analytes of interest discussed herein.

The sample, or the processed sample, may be purified prior to analysisby mass spectrometry. Such purification, or sample clean-up, refers to aprocedure that enriches of one or more analytes of interest relative toone or more other components of the sample. Typically, one or moremethods including, without limitation, liquid chromatography, HPLC,UHPLC, precipitation, dialysis, affinity capture, electrophoresis, orother suitable methods known in the art, are used for the purification.

Various methods have been described involving the use of HPLC for sampleclean-up prior to mass spectrometry analysis. See, e.g., Taylor et al.,Therapeutic Drug Monitoring 22:608-12 (2000) (manual precipitation ofblood samples, followed by manual C18 solid phase extraction, injectioninto an HPLC for chromatography on a C18 analytical column, and MS/MSanalysis); and Salm et al., Clin. Therapeutics 22 Supl. B:B71-B85 (2000)(manual precipitation of blood samples, followed by manual C18 solidphase extraction, injection into an HPLC for chromatography on a C18analytical column, and MS/MS analysis). One of skill in the art canselect HPLC instruments and columns that are suitable for use in theinvention. The chromatographic column typically includes a medium (i.e.,a packing material) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles. The particlesmay include a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the analytes of interest. Onesuitable bonded surface is a hydrophobic bonded surface such as an alkylbonded surface. Alkyl bonded surfaces may include C-4, C-8, or C-18bonded alkyl groups, preferably C-18 bonded groups. The chromatographiccolumn includes an inlet port for receiving a sample and an outlet portfor discharging an effluent that includes the fractionated sample. Forexample, a test sample may be applied to the column at the inlet port,eluted with a solvent or solvent mixture, and discharged at the outletport. In another example, more than one column may be used wherein atest sample may applied to a first column (e.g., a clean-up column suchas a Cyclone P column or the like) at the inlet port, eluted with asolvent or solvent mixture onto a second column (e.g., an analyticalcolumn such as a Hypersil Gold PFP, Accucore PFP™, Halo or the like),and eluted with a solvent or solvent mixture from the second column tothe outlet port. Different solvent modes may be selected for eluting theanalytes. For example, liquid chromatography may be performed using agradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode.

Recently, high turbulence liquid chromatography (“HTLC”), also calledhigh throughput liquid chromatography, has been applied for samplepreparation prior to analysis by mass spectrometry. See, e.g., Zimmer etal., J. Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos.5,968,367; 5,919,368; 5,795,469; and 5,772,874, each of which is herebyincorporated by reference in its entirety. Traditional HPLC analysisrelies on column packings in which laminar flow of the sample throughthe column is the basis for separation of the analyte of interest fromthe test sample. The skilled artisan will understand that separation insuch columns is a diffusional process. In contrast, it is believed thatturbulent flow, such as that provided by HTLC columns and methods, mayenhance the rate of mass transfer, improving the separationcharacteristics provided. In some embodiments, high turbulence liquidchromatography (HTLC), alone or in combination with one or morepurification methods, may be used to purify the analyte(s) of interest.In such embodiments samples may be extracted using an HTLC extractioncartridge which captures the analyte, then eluted and chromatographed onan HPLC or other column prior to ionization. Because the steps involvedin these two column purification steps can be linked in an automatedfashion, the requirement for operator involvement during thepurification of the analyte can be minimized

The terms “mass spectrometry” or “MS” as used herein refer to methods offiltering, detecting, and measuring ions based on their mass-to-chargeratio, or “Dale.” In general, one or more molecules of interest, such avitamin D metabolites, are ionized and the ions are subsequentlyintroduced into a mass spectrographic instrument where, due to acombination of magnetic and electric fields, the ions follow a path inspace that is dependent upon mass (“m” or “Da”) and charge (“z” or “e”).

The mass spectrometer 150 will include an ion source for ionizing thefractionated sample and creating charged molecules for further analysis.For example ionization of the sample may be performed by electrosprayionization (ESI). Other ionization techniques include, but are notlimited to, atmospheric pressure chemical ionization (ACPI),photoinonization, electron impact ionization, chemical ionization,fastatom bombardment (FAB)/liquid secondary ion mass spectrometry (LSIMS),matrix assisted laser desorption ionization (MALDI), field ionization,field desorption, thermospray/plasmaspray ionization, and particle beamionization. The skilled artisan will understand that the choice ofionization method can be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., Da/e). Suitable analyzers for determiningmass-to-charge ratios include quadrupole analyzers, ion traps analyzers,Fourier transform ion cyclotron resonance (FTICR) mass analyzers,electrostatic trap analyzers, magnetic sector analyzers andtime-of-flight analyzers. The ions may be detected by using severaldetection modes. For example, selected ions may be detected (i.e., usinga selective ion monitoring mode (SIM)), or alternatively, ions may bedetected using selected reaction monitoring (SRM) or multiple reactionmonitoring (MRM) (MRM and SRM are essentially the same experiment.).Ions can also be detected by scanning the mass spectrometers to detectall the precursor ions simultaneously or all the products ions of aspecific precursor ion simultaneously or both.

In one embodiment, the mass-to-charge ratio is determined using aquadrupole analyzer. For example, in a “quadrupole” or “quadrupole iontrap” instrument, ions in an oscillating radio frequency fieldexperience a force proportional to the DC potential applied betweenelectrodes, the amplitude of the RF signal, and Da/e. The voltage andamplitude can be selected so that only ions having a particular Da/etravel the length of the quadrupole, while all other ions are deflected.Thus, quadrupole instruments can act as a “mass filter,” a “massseparator” or an ion lens for the ions injected into the instrument.

One can often enhance the resolution of the MS technique by employing“tandem mass spectrometry” or “MS/MS” for example via use of a triplequadrupole mass spectrometer. In this technique, a first, or parent, orprecursor, ion generated from a molecule of interest can be filtered inan MS instrument, and these precursor ions subsequently fragmented toyield one or more second, or product, or fragment, ions that are thenanalyzed in a second MS procedure. By careful selection of precursorions, only ions produced by certain analytes are passed to thefragmentation chamber (e.g., a collision cell), where collision withatoms of an inert gas to produce these product ions. Because both theprecursor and product ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquecan provide an extremely powerful analytical tool. For example, thecombination of ion selection or filtration and subsequent fragmentationcan be used to eliminate interfering substances, and can be particularlyuseful in complex samples, such as biological samples.

For example, a flow of liquid solvent from a chromatographic column,possibly containing one or more analytes of interest, enters the heatednebulizer interface of a LC-MS/MS analyzer and the solvent/analytemixture is converted to vapor in the heated tubing of the interface.Ions derived from the analytes of interest may be formed in the liquidphase and subsequently ejected into the gas phase by nebulization in theESI source or by reactions between neutral analytes and reactive ions(e.g., ammonium ions) as the analytes enter the gas phase.

The ions pass through the orifice of the instrument and enter the firstquadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowingselection of ions based on their mass to charge ratio (Dale). Quadrupole2 (Q2) is the collision cell, where ions are fragmented. The firstquadrupole of the mass spectrometer (Q1) selects for molecules with themass to charge ratios (“Dale”) of ions specific to each analyte ofinterest to be analyzed. Ions with the correct Dale ratios of theselected analyte of interest are allowed to pass into the collision cell(Q2), while unwanted ions with any other Dale are ejected from orcollide with the sides of the quadrupole (Q1) and are eliminated. Ionsentering Q2 collide with neutral gas molecules (e.g., argon) andfragment. This process is called Collision Activated Dissociation (CAD).The fragment ions generated are passed into quadrupole 3 (Q3), where thefragment ions of the chosen analyte of interest are selected while otherions are eliminated. As ions collide with the detector they produce apulse of electrons that are converted to a digital signal.

Mass spectrometry instruments can vary slightly in determining the massof a given analyte. Thus, the term “about” in the context of mass of anion or the Dale of an ion refers to +/−0.2, +/−0.3, or +/−0.5 atomicmass units or Daltons (Da).

The acquired data is relayed to a computer, which plots voltage versustime. The resulting mass chromatograms are similar to chromatogramsgenerated in traditional HPLC methods. Concentrations of the analytes ofinterest may be determined by calculating the area under the peaks inthe chromatogram. The concentration of the analyte or analytes ofinterest in the sample is typically determined either by comparing thearea under the peaks to a calibration curve or comparing the ratio ofinternal standards (e.g., deuterated 25-hydroxyvitamin D₃) to testsamples.

III. Kits for LC-MS Purification and Detection of Analytes from a BroadRange of Chemical Classes

In one embodiment, a kit for performing LC-MS of a sample containing atleast one analyte of interest is disclosed. The kit includes reagentsfor purifying, eluting and analyzing a plurality of analytes of interestusing an LC-MS system, and a protocol that includes instructions forpurifying and analyzing the plurality of analytes of interest havingpartition coefficients (logP) ranging from about −1 to about 6 using theLC-MS system.

Suitable examples of analytes of interest that can be purified, elutedand analyzed using the methods systems and kits described hereininclude, but are not limited to, vitamin D, steroid hormones,immunosuppressants, chemotherapeutics, tricyclic antidepressants, azoleantifungals, anti-epileptics, anti-retrovirals, opiates and/or opioids,drugs of abuse, barbiturates, benzodiazepines, metabolites thereof, andcombinations thereof.

In one embodiment, the protocol may include instructions relating tosample handling and preparation, liquid chromatography conditions (e.g.,flow rates, gradients, columns, temperature, etc.), composition ofbuffers for LC-MS, mass spectrometer settings (e.g., spray voltage,desolvation temperature, sheath gas pressure, voltage and scan settingsfor the mass analyzer(s), etc.).

The protocol included in the kit can also include instructions forpurifying and analyzing the analytes having logPs ranging from about−1.2 to about 6 such that a diagnostic quality standard is satisfied foreach analyte in the logP range from about −1.2 to about 6.

As used herein, the term “diagnostic quality standard” refers to ananalytical performance aspect that includes achieving reproduciblequantitation for each analyte within specified detection limits, withspecified precision and substantially no carry-over from sample tosample.

As used herein, “detection limit(s)” refers to a limit that at leastspans the reference range for a given analyte, the lower limit ofquantitation being no higher than the low end of the reference range andthe upper limit of quantitation being no lower than the upper end of thereference range, wherein the reference range is relevant to a particularapplication including, without limitation, clinical, industrial andenvironment applications.

Lower and upper clinical reference ranges for many of the compoundsdiscussed herein are listed in Table 2 (below) in the “Low” and “High”columns, respectively. Additional examples of lower and upper referenceranges for most clinically relevant analytes of interest can be found inTietz Textbook of Clinical Chemistry and Molecular Diagnostics (Burtiset al, Saunders; 4^(th) edition (1 Jul. 2005), ISBN: 0721601892), theentirety of which is incorporated herein by reference. Note, however,that Table 2 and Tietz relate to clinical reference standards as theyare now recognized in clinical practice; however, detection limits andclinical reference ranges are continuously evolving. Note also, thatclinical reference ranges may vary depending on the standard of care inmedical practice and or regulatory bodies in different countries (e.g.,the United Stated vs. France). Likewise, because different age groups,genders, and ethnic groups are known to respond differently to somedrugs, clinical reference ranges can also vary depending on the groupsbeing tested.

Note also that the lower and upper ends of the clinical reference rangedo not necessarily represent the limit of detection and the limit ofquantitation. For example, the limits of detection and quantitation maybe as much as several orders of magnitude lower than the lower clinicalreference range. Likewise, the upper range of concentration that can beaccommodated by the methods and apparatuses described herein may be muchhigher than the upper clinical reference range.

As used herein, the term “precision” refers to reproducibility ofspecific measurements as measured by a coefficient of variation forrepeat measurements of a single sample. Precision is customarilydetermined as a step of assay development, wherein coefficients ofvariation are derived from repeat measurements on the same day, overdifferent days, etc. Precision requirements vary from one analyte toanother depending on current medical practice; however it is generallyunderstood that coefficients of variation below 15% are preferred.

As used herein, the term “substantially no carry-over” refers to asituation where essentially no sample contamination from previousanalysis remains in the system (i.e., the LC-MS system) betweenanalyses. The extent of carry-over in analytical instruments can beassessed in a number of ways. One method involves running a number ofblank samples in order to establish a clear baseline, running a numberof samples having a high concentration of an analyte of interest (e.g.,25OH vitamin D₃), followed by assaying blank samples again to ensure areturn to baseline. Carry-over can also be assessed by seriallyanalyzing low- and high-concentration control samples (e.g., samplesrepresenting the lower and upper limits of quantitation). In oneexample, “carry-over” is no larger than 0.1% of the blank immediatelyafter analyzing a sample having a high concentration of an analyte ofinterest. In another example, “carry-over” is no larger than 20% of thelower limit of quantitation (LLOQ) after analyzing a sample at the upperlimit of quantitation (ULOQ).

In one embodiment, the kit further includes either a single liquidchromatography column (e.g., a single analytical liquid chromatographycolumn) or a single set of liquid chromatography columns (e.g., a singlesample clean-up liquid chromatography column that, when in use, is influid communication with and upstream of the single analytical liquidchromatography column) capable of effecting separation of a variety ofanalytes from a biological matrix and/or from a “neat” sample, at leastone liquid chromatography buffer solution containing a source ofammonium ions, and at least one quality-control standard or internalstandard for tracking at least one of separation, ionization,fragmentation, or detection of at least one analyte of interest.

Chromatographic columns typically include an inlet port for receiving asample and an outlet port for discharging an effluent that includes thefractionated sample. For example, a test sample may applied to thecolumn at the inlet port, eluted with a solvent or solvent mixture, anddischarged at the outlet port. In another example, more than one columnmay be used wherein a test sample may applied to a first column (e.g., aclean-up column) at the inlet port, eluted with a solvent or solventmixture onto a second column (e.g., an analytical column), and elutedwith a solvent or solvent mixture from the second column to the outletport.

Many types of HPLC and UHPLC columns are commercially available and canbe selected based on various criteria known to persons having ordinaryskill in the art. For example, commercially available HPLC and UHPLCcolumns include normal-phase (polar stationary phases and non-polarmobile phases), reverse-phase (the stationary phase is non-polar and themobile phase is polar), ion-exchange (charged species on the stationaryphase and charged species in the mobile phase), and affinitychromatography (based on specific interactions in a lock-and-keyparadigm between analytes and matrix-bound ligands). In one embodiment,the liquid chromatography is reverse-phase. Suitable reverse phasecolumns include, but are not limited to, C-4, C-8, C-18, Hypersil GoldPFP, Accucore PFP™, Halo and the like.

In one embodiment, the one aqueous mobile phase buffer solution and theone substantially non-aqueous mobile phase buffer solution include asource of ammonium ions. Acceptable examples of ammonium ion sourcesinclude ammonium formate and/or ammonium acetate. In one embodiment,ammonium formate and/or ammonium acetate may be included in the mobilephase buffer in an amount ranging from about 2 mM to about 10 mM, orabout 4 mM to about 8 mM, or about 10 mM. Lower or higher amounts ofammonium formate and/or ammonium acetate may be used (e.g., about 0.1 mMto about 20 mM, 50 mM, or 100 mM), but below approximately about 2 mMthe numbers of ammonium ions in solution and/or the gas phase may beinsufficient to promote ionization of the analytes of interest; amountsabove approximately about 10 mM increase the risk that charge-chargerepulsion of ammonium ions in solution and/or the gas phase can produceartifacts and reduce sensitivity.

Ammonium formate and ammonium acetate are acceptable sources of ammoniumions for use in LC-MS. Ammonium formate's pKa is lower than ammoniumacetate's and, as such, the analytes of interest will be expected to bemore fully ionized in the buffer solution, which may be desirable insome instances. Both ammonium formate and ammonium acetate areacceptable because they are sources of volatile ions and are notexpected to interfere with mass spectrometry results. In contrast,ammonium chloride, which is also a source of ammonium ions, is generallyconsidered to be unacceptable for use in LC-MS because the chloride isnot volatile and would therefore be expected to foul the massspectrometer.

In one embodiment, the one aqueous mobile phase buffer solution includesan aqueous solution (e.g., water, ammonium formate, and formic acid). Inone embodiment, the one substantially non-aqueous mobile phase buffersolution includes a non-aqueous solution (e.g., methanol, ammoniumformate, and formic acid). Other organic phases that can be usedinclude, but are not limited to, acetonitrile, ethanol, isopropanol, andcombinations thereof. Samples containing one or more analytes ofinterest can be loaded and washed with aqueous buffer and eluted in anaqueous to non-aqueous gradient or isocratically (e.g., with 100%non-aqueous buffer). In one embodiment, the reagents included in the kitcan further include a chromatography wash buffer configured to wash thesystem in between samples. In one example, the wash buffer may includeisopropyl alcohol (“IPA”), acetonitrile (“ACN”) and acetone. In anotherembodiment, the wash buffer may be a high pH aqueous wash buffer ofabout 8.0 to 10 pH. Optionally, the wash buffer may contain a chelatingagent such as, but not limited to, EDTA.

IV. Methods for Purifying and Analyzing Analytes having a Wide Range ofHydrophobicities

Referring now to FIG. 2, a method 200 for purifying and analyzinganalytes having a wide range of hydrophobicities using liquidchromatography-mass spectrometry (LC-MS) is illustrated. The illustratedmethod 200 includes an action 210 of providing at least a first analytefrom a first chemical class and an action 220 of providing at least asecond analyte of interest from a second chemical class that isdifferent from the first chemical class. The first and second analytesof interest have log partition coefficients (logP) spanning a range fromabout −1.2 to about 6. The method further includes an action 230 ofproviding an LC-MS system that includes a single analytical columnconfigured to at least partially purify each of the first and secondanalytes of interest, a mass spectrometer (MS), a single aqueous mobilephase buffer solution, and a single substantially non-aqueous mobilephase buffer solution. In one embodiment, the mobile phase buffersolutions each contain a source of ammonium ions. The method furtherincludes an action 240 of purifying and eluting the first analyte ofinterest for analysis in the MS, and an action 250 of purifying andeluting the second analyte of interest for analysis in the MS.

In one embodiment, the first analyte of interest has a first logP andthe second analyte of interest has a second logP that is different fromthe first logP. In one embodiment, the first and second logPs may beseparated by at least about 4.5 logP units. Suitable examples ofanalytes of interest that can be purified and analyzed using method 200include, but are not limited to, vitamin D, steroid hormones,immunosuppressants, chemotherapeutics, tricyclic antidepressants, azoleantifungals, anti-epileptics, anti-retrovirals, opiates and/or opioids,drugs of abuse, barbiturates, benzodiazepines, metabolites thereof, andcombinations thereof. These analytes have logPs ranging from about −1.2(busulfin) to about 6 (25-OH vitamin D₂) (see, e.g., Table 1).

In one embodiment, the actions 210 and 220 can include providing two ormore analytes selected from a first group and a second group, whereinthe log partition coefficients (logP) of the first and second groups areseparated by at least about 4.5 logP units. In another embodiment, theactions 210 and 220 can include providing a first analyte and a secondanalyte selected from the group consisting of vitamin D, steroidhormones, immunosuppressants, chemotherapeutics, tricyclicantidepressants, azole antifungals, anti-epileptics, anti-retrovirals,opiates and/or opioids, drugs of abuse, barbiturates, benzodiazepines,or a metabolite thereof, wherein the log partition coefficients (logP)of the first and second analytes are separated at least about 4.5 logPunits. In yet another embodiment, the actions 210 and 220 can includeproviding a first analyte, a second analyte, and a third analyte,wherein the first analyte has a log partition coefficient (logP) in arange of about −1.2 to about 0, the second analyte has a logP in a rangefrom about 0 to about 5, and the third analyte has a logP greater thanabout 5 and less than or equal to about 6.

Purifying and analyzing the first analyte of interest (240) followed bypurifying an analyzing the second analyte of interest (250) isaccomplished by varying at least one LC-MS system parameter selectedfrom the group consisting of flow rate, composition of the mobile phasebuffer solutions, a ratio of the aqueous mobile phase buffer solution tothe non-aqueous mobile phase buffer solution, a gradient varying ratiosof the aqueous and non-aqueous mobile phase buffer solutions, ionizationvoltage, desolvation temperature, lens amplitude, collision gastemperature, collision gas pressure, collision energy, and combinationsthereof. Notably, purifying and analyzing the first analyte of interest(240) followed by purifying an analyzing the second analyte of interest(250) does not involve having to alter column or buffer selection.

In one embodiment, the additive included in the mobile phase buffersincludes a source of ammonium ions. In one embodiment, the source ofammonium ions is ammonium formate or ammonium acetate. In oneembodiment, the additive in the aqueous and non-aqueous mobile phasebuffers includes about 2 mM to about 15 mM ammonium, about 2 mM to about10 mM ammonium, or about 10 mM ammonium in the form of ammonium formate,ammonium acetate, or another suitable source of ammonium ions.

In one embodiment, the liquid chromatography system provided in action230 is a high-performance liquid chromatography (“HPLC”) system or anultra high-performance liquid chromatography (“UHPLC”) system. In oneembodiment, the liquid chromatography system provided in action 230includes a single sample clean-up liquid chromatography column (e.g., aCyclone P column) and an analytical liquid chromatography column (e.g.,e.g., a Hypersil Gold PFP, Accucore PFP™, Halo column). The sampleclean-up liquid chromatography column and the analytical liquidchromatography column are in fluid communication with one another withthe sample clean-up liquid chromatography column being upstream of theanalytical liquid chromatography column. In one embodiment, the massspectrometer includes an electrospray ionization (ESI) and/or an APCIion source.

Ammonium ions in the form of ammonium formate, ammonium acetate, oranother suitable source of ammonium ions may participate in theformation ions specific to each analyte of interest. For some analytes,ammonium ions may promote the formation of protonated ion form of theanalyte of interest by donating a proton to the analyte either in theliquid phase or the gas phase. For some other analytes, (e.g., steroids)ammonium ions may promote the formation of ammoniated ions. In general,protonated and/or ammoniated molecular ions are preferred over waterloss ions common in some ionization techniques because the analytes ofinterest have many possible routes for water loss, which can make waterloss ions difficult to track in the mass spectrometer.

In one embodiment, the LC-MS system provided in action 230 furtherincludes a sample handing device configured to manage a plurality ofsamples and a control system coupled to the LC-MS system and configuredto control or vary at least one of a priority of analysis for the firstsample and the second sample, flow rate, a ratio of the aqueous mobilephase buffer solution to the non-aqueous mobile phase buffer solution, agradient varying ratios of the aqueous and non-aqueous mobile phasebuffer solutions, ionization voltage, desolvation temperature, lensamplitude, collision gas temperature, collision gas pressure, andcombinations thereof. The sample handling device and the control systemare discussed in greater detail elsewhere in this application in thecontext of the system.

Referring now to FIG. 3, another method 300 for detecting and/orquantifying analytes having a wide range of partition coefficients usingliquid chromatography-mass spectrometry (LC-MS) is illustrated. Themethod 300 includes an action 310 of providing an LC-MS systemconfigured for purifying and analyzing a plurality of analytes ofinterest having partition coefficients (logP) spanning a range fromabout −1 to about 6, an action 320 of selecting a sample containing aselected analyte of interest, an action 330 of selecting an analysisprotocol based on the selected analyte of interest, and an action 340 ofpurifying, eluting, and analyzing the selected analyte of interest byLC-MS.

In one embodiment, the action 300 includes providing a single analyticalliquid chromatography column of a liquid chromatography-massspectrometry (LC-MS) system, one aqueous mobile phase buffer solution ofthe LC-MS system, and one substantially non-aqueous mobile phase buffersolution of the LC-MS. The LC-MS system is configured for purifying andanalyzing a plurality of analytes having a log partition coefficients(logP) spanning a range of about −1.2 to about 6 by varying at least oneLC-MS system parameter. LC-MS system parameters include, but are notlimited to, mobile phase buffer flow rate, a ratio of an aqueous mobilephase buffer solution to a non-aqueous mobile phase buffer solution, agradient varying ratios of the aqueous and non-aqueous mobile phasebuffer solutions, ionization voltage, desolvation temperature, electrodevoltage, collision gas temperature, collision gas pressure, collisionenergy, and combinations thereof.

EXAMPLES Example 1 Purification and Analysis of 25-OH Vitamin D₂ and25-OH Vitamin D₃

25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ were purchased from Sigma(St. Louis, Mo.). A 2 mg/mL stock solution was made by dissolving25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ in methanol. All otherconcentrations were made by serial dilutions into methanol (neat) orstripped serum (matrix) such as charcoal-stripped fetal bovine serum(Sigma Aldrich, Cat. No. F6765) or antibody-stripped human serum.Standards and QCs were made in stripped serum. Standards were made witha range of 1-300 ng/mL and QCs were made 2, 120 and 240 ng/mL.Hexadeuterated 25-hydroxyvitamin D₃ (d6-25-OH D3) was purchased fromMedical Isotopes (Cat. No. D2831) and used as an internal standard (IS).A 1 mg/mL IS stock solution was made in methanol and diluted to 140ng/mL with methanol for the working IS stock solution. All stock andworking solutions were stored at −80° C.

Samples were prepared by adding 200 μL of working internal standard to100 μL of sample followed by vortex mixing for 15 sec at max speed andcentrifugation at 4000 rcf for 2 min. 150 μL of supernatant was thentransferred into autosampler vials for analysis.

HPLC was performed with Thermo Scientific Transend TX system (ThermoFisher Scientific) using a 0.5×50 mm Cyclone P column (Thermo FisherScientific) for on-line sample clean-up and a 2.1×50 mm, 1.9 μm HypersilGold PFP or 2.1×50 mm, 2.6 μm Accucore PFP™ analytical column (ThermoFisher Scientific). This system is a dual column system that can performUltra High Pressure Liquid Chromatography (UHPLC) and utilizes TurboFlowtechnology to perform on-line clean-up. The mass spectra were acquiredon a Thermo Scientific Vantage triple quadrupole mass spectrometer(Thermo Fisher Scientific). Mobile phase A was 10 mM ammonium formatewith 0.01% formic acid in water. Mobile phase B was 10 mM ammoniumformate with 0.01% formic acid in methanol. Mobile phase C was 45:45:10isopropanol:acetonitrile:acetone that is used to wash the columns

10 μL of sample was injected onto the turbulent flow chromatography(“TFC”) column with 80% mobile phase A at 1.5 mL/min Large molecules,such as proteins, are washed to waste while small molecules (>1000 Da)are retained on the column. Once the sample has been separated from mostof the biological matrix, the valves switch and the sample is elutedfrom the TFC column with 100% mobile phase B at 0.2 mL/min. The flowfrom the TFC column is teed to a second UHPLC pump flowing 80% mobile Aat 0.5 mL/min. The mixed flow from both pumps reduces the amount oforganic seen by the analytical column such that the analyte of interestis focused at the head of the analytical column. Once the analyte ofinterest is transferred to the analytical column, the valves areswitched again, isolating the two columns from each other. The TFCcolumn is washed and equilibrated for the next sample and a 20-100%mobile phase B gradient is run on the analytical column to elute theanalyte of interest into the mass spectrometer for analysis.

The mass spectrometer parameters are as follows: Spray voltage 5000,Vaporizer temperature 400, sheath gas pressure 60, aux gas pressure 35,capillary temperature 199, S-lens amplitude 55. Full scan Q1 data wasacquired to look at the relative ion abundances of the methods testedand Selective Reaction Monitoring (SRM) was used for quantitativecomparison.

The SRM transitions used were as follows; 25-hydroxyvitamin D₃: 401.352parent, 91.122, 105.133, 159.139, 365.425 product ions.25-hydroxyvitamin D₂: 91.089, 95.158, 105.104, 159.149 product ions.d₆-25-OH D₃: 407.380 parent, 107.115, 133.105, 147.199, 159.190 productions. Quadrupole 1 (Q1) (full width at half maximum, FWHM) was set at0.2 and quadrupole 3 (Q3) (FWHM) was set at 0.7. Scan width (Dale) 0.01,scan time (s) 0.05. Collision gas pressure was set at 1.5 mTorr.

Comparison of full scan data for the [M+H]+ ion using ammonium formatein the mobile phase verses the [M+H—H₂O]+ for Vitamin D₂, for example,using formic acid in the mobile phase are shown in the FIGS. 4A-4B. Notethat the addition of an ammonium source significantly increases thesensitivity of detection and significantly improves the signal-to-noiseratio. Note also that the [M+H]+ ion is not detected without ammoniumions.

More important to the quantitation using MS/MS is the comparison of theSRM data from using the [M+H]+ and [M+H—H₂O]+ as the precursor ions.Those comparisons are shown for 25-OH D₂ in FIGS. 5A and 5B. It is clearthat the use of the [M+H]+ ion formed by the presence of ammonium ionsin the mobile by electrospray has higher sensitivity then using the[M+H—H₂O]+ ion by APCI. The data in FIGS. 5A and 5B illustrate thatdetection and quantitation of 25-hydroxyvitamin D₂ with the [M+H]+ ionfrom ESI is about 3.5 times more sensitive than detection with the[M+H—H₂O]+ from APCI.

Further discussion of LC-MS purification and analysis of vitamin Dmetabolites can be found in U.S. Prov. Pat. App. Ser. No. 61/408,385entitled “LC-MS SEPARATION AND DETECTION OF VITAMIN D METABOLITES” filed29 Oct. 2010 with inventors Joseph L. Herman and Dayana Argoti, theentirety of which is incorporated herein by reference.

Example 2 Purification and Analysis of Analytes of Interest in NeatSamples and Serum

FIGS. 6-15 depict MRM chromatograms illustrating purification andanalysis of 40 separate analytes of interest in neat samples on aCyclone P clean-up column followed by a Hypersil Gold PFP analyticalcolumn. Isocratic runs of varying amounts of organic mobile phase wereused to characterize the retentive properties of each analyte. Based onthe these results the final mobile phases chosen were Solution A: 10 mMammonium formate, 0.01% formic acid, water and Solution B: 10 mMammonium formate, 0.01% formic acid, methanol. FIGS. 6-15 illustratethat baseline separation of compounds having hydrophobicities rangingacross about 7 logPs can be achieved using the methods, systems, andkits described herein. It was unexpected that compounds so widelyranging in logPs could be separated using the methods systems and kitsdescribed herein. The only parameters that were varied across thevarious compounds were LC run parameters (e.g., flow rate, ratios of onebuffer to another, temperature, and the like) and MS system parameters(e.g., ionization voltage, desolvation temperature, and the like).Columns and buffers were not changed.

FIGS. 16-20 depict MRM chromatograms illustrating purification andanalysis of a number of separate analytes of interest ranging in logPfrom about 0.1 (norcodeine) to about 5.01 (everolimus). The analytes ofinterest were dissolved in antibody stripped human serum and purified ona Cyclone P clean-up column followed by an Accucore PFP™ analyticalcolumn. Based on these results, the final mobile phases identified above(i.e., Solution A: 10 mM ammonium formate, 0.01% formic acid, water andSolution B: 10 mM ammonium formate, 0.01% formic acid, methanol) wereshown to be effective under these conditions. For the analytes ofinterest illustrated in FIGS. 16-20, no recovery or matrix effects(i.e., interference from the matrix as compared to “neat” samples) weredetected. In addition, comparing FIGS. 11 and 20, for example, it can beseen that the antibody stripped human serum, Cyclone P clean-up column,and Accucore PFP™ analytical column can provide better separation thanseen with “neat” samples separated with a Cyclone P clean-up columnfollowed by a Hypersil Gold PFP analytical column As with FIGS. 6-15,the only parameters that were varied across the various compoundsillustrated in FIGS. 16-20 were LC run parameters (e.g., flow rate,ratios of one buffer to another, temperature, and the like) and MSsystem parameters (e.g., ionization voltage, desolvation temperature,and the like). Columns and buffers were not changed.

Referring now to Table 2, additional data regarding the analytes ofinterest are illustrated. The compounds listed in table 2 can beanalyzed by LC-MS for a variety of clinical and drug monitoring purposesusing the methods, systems, and kits described herein. The compoundslisted in Table 2 have logPs ranging from about −1.2 (busulfan) to about6 (25OH vitamin D₂), which represents a difference in hydrophobicityover slightly more than about seven orders of magnitude.

Table 2 also shows the lower and upper clinical reference ranges formany of the compounds listed in Table 2 in the “Low” and “High” columns,respectively, and the approximate limit of quantitation (LOQ) using themethods, systems, apparatuses, and kits described herein. The lower andupper clinical reference ranges represent the dynamic range for each ofthe compounds expected for the compounds in a clinical context. Inessentially all cases, however, the limits of detection and the LOQs aremuch lower than the lower clinical reference range.

The LOQs listed in Table 2 range from a high of about 50 ng/mL for theamphetamines to a low of about 10 pg/mL for some of the steroids. Thisis at least as good as and in some cases far better than the LOQs forcurrently practiced methods that have been optimized for each analyte.However, methods that have been optimized for each separate analytecannot be adapted for purification and analysis of analytes havingdifferences in hydrophobicity over slightly more than about seven ordersof magnitude. That is, a typical LC-MS method that is optimized for ananalyte or a class of analytes such as vitamin D metabolites is cangenerally only be used for purification and analysis of that particularanalyte or class of analytes. In contrast, the methods, kits, systems,and apparatuses described herein are useful for purification andanalysis of analytes having differences in hydrophobicity over slightlymore than about seven orders of magnitude. In addition, it was observed,quite unexpectedly, that electrospray ionization (ESI) improvedsensitivity relative to atmospheric pressure chemical ionization (APCI),for many of the analytes tested using the methods systems and kitsdescribed herein (Table 2). APCI is recognized as the accepted method ofionization for many common analytes of interest.

TABLE 2 Endocrinology Vitamin D logP Low High Units Approx [LOQ]ESI/APCl 25OH vitamin D2 5.69 10 65 ng/mL 10 ng/mL 8.81 25OH vitamin D35.61 10 ng/mL 3.32 logP Low High Units Approx [LOQ] ESI/APCI SteroidsTestosterone 4.44 0.01 10 ng/mL 50 pg/mL 16.4 Cortisol (hydrocortisone)1.26 1 70 μg/day 50 pg/mL 54.5 Cortisone 1.58 50 pg/mL 35.4 Progesterone3.58 10 pg/mL Hydroxyprogesterone 3.52 10 pg/mL Prednisone 2.07 10 pg/mLAndrostenedione 2.93 10 pg/mL Therapeutic Drug MonitoringImmunosuppressants Tacrolimus 3.3 1 30 ng/mL 50 pg/mL 21.7 Everolimus5.01 0.3 200 ng/mL 50 pg/mL 29.2 Sirolimus 4.3 0.3 200 ng/mL 50 pg/mL27.3 Cyclosporine A 4.12 5 1000 ng/mL 500 pg/mL 9.52 ChemotherapeuticsMethotrexate 0.94 1 1000 ng/mL 500 pg/mL Busulfan −1.15 5 2000 ng/mL 1ng/mL 5-Fluorouracil −0.9 0.01 1 ng/mL Docetaxel 2.4 1 1000 ng/mL 500pg/mL Pain management & Drugs of Abuse NIDA 5 Phencyclidine 4.14 0.5ng/mL Benzoylecgonine 1.64 5 2500 ng/mL 0.05 ng/mL 12.2 Cocaine 1.910.05 ng/mL 16 Delta9-THC 5.53 5 ng/mL 0.75 11-norDelta-9-THC-COOH 4.6 101250 ng/mL 5 ng/mL 0.53 Amphetamines Amphetamine 1.7 51 5000 ng/mL 50ng/mL Methamphetamine 2.2 51 5000 ng/mL 50 ng/mL MDMA 1.98 51 5000 ng/mL50 ng/mL MDEA 2.31 51 5000 ng/ml 50 ng/mL MDA 1.46 51 5000 ng/ml 50ng/ml Opiates/Opioids Hydromorphone 1 5 1000 ng/mL 2 ng/mLNorhydrocodone 0.89 2 ng/mL Norcodeine 1.07 2 ng/mL Morphine 1.73 251000 ng/mL 20 ng/mL Hydrocodone 1.27 2 1000 ng/mL 2 ng/mL Codeine 1.45 21000 ng/mL 2 ng/mL Noroxycodone 0.1 2 ng/mL Oxymorphone 0.21 5 1000ng/mL 20 ng/mL Dihydrocodeine 1.63 5 1000 ng/mL 0.2 ng/mL Oxycodone 0.485 1000 ng/mL 2 ng/mL 6-MAM 1.81 2 ng/mL Tapentadol 3.43 0.5 ng/mL 7.1Norfentanyl 3.94 0.5 200 ng/mL 0.05 ng/mL 10.3 Fentanyl 4.59 0.5 200ng/mL 0.05 ng/mL 3.3 Tramadol 2.53 5 ng/mL Methadone 4.55 5 ng/mL 14.5Metoprolol 2.18 0.05 ng/mL 8.7

Example 3 Performance of Immunosuppressant Drugs, 25-Hydroxy Vitamin D₂and D₃, and Chemotherapeutics in Biological Matrix Solution

FIGS. 21-26 illustrate the performance of immunosuppressant drugs,25-hydroxy vitamin D₂ and D₃, and the chemotherapeutic drugs docetaxceland busulfin in matrix (e.g., blood or serum). No recovery or matrixeffects (i.e., interference from the matrix as compared to “neat”samples) were detected for the immunosuppressant drugs, 25-hydroxyvitamin D₂ and D₃, docetaxel or busulfan in biological matrix solution.It is interesting to note that busulfin (logP≈−1.15) and 25-hydroxyvitamin D₂ (logP≈5.69) represent the lower and upper logP ranges foranalytes tested using the methods described herein. It was unexpectedthat compounds as different as busulfin and 25-hydroxy vitamin D₂ couldbe separated using the methods systems and kits described herein. Theonly parameters that were varied from busulfin to 25-hydroxy vitamin D₂were LC run parameters (e.g., flow rate, ratios of one buffer toanother, temperature, and the like) and MS system parameters (e.g.,ionization voltage, desolvation temperature, and the like). Columns andbuffers were not changed.

Example 4 Method Details for Purification and Analysis of the Analytesof Interest

Tables 3-11 illustrate various method details for purification andanalysis of the various analytes of interest discussed herein.

TABLE 3 System set-up/configuration Data Window Start  20 Data WindowLength 359 Column One Cyclone P Column Two Hypersil Gold PFP or AccucorePFP Method Comment Loading Pump Quaternary Pump Solvent A 10 mMAmmFormt/0.01% FA H₂O Solvent B 10 mM AmmFormt/0.01% FA 95:5 ACN:H₂OSolvent C 45/45/10 IPA/ACN/Acetone Solvent D 10 mM AmmFormt/0.01% FAMeOH Eluting Pump Quaternary Pump Solvent A 10 mM AmmFormt/0.01% FA H₂OSolvent B 10 mM AmmFormt/0.01% FA 95:5 ACN:H₂O Solvent C 45/45/10IPA/ACN/Acetone Solvent D 10 mM AmmFormt/0.01% FA MeOH

TABLE 4 LC Gradient details for ISDs, Steroids and Drugs of AbuseLoading Eluting Step Start Sec Flow Grad % A % B % C % D Tee Loop FlowGrad % A % B % C % D Comment 1 0:00 25 1.50 Step 100 ~~~~ out 0.50 Step100 Sample clean-up 2 0:25 5 0.10 Step 100 ~~~~ out 0.35 Step 100 Lowerflow rate 3 0:30 60 0.10 Step 100 T in 0.35 Step 100 Transfer 4 2:00 601.50 Step 100 T in 0.35 Ramp 100 Loop fill/Grad Elute 5 2:15 30 1.50Step 100 ~~~~ in 0.50 Step 100 Loop fill/Grad Elute 6 2:30 30 1.50 Step100 ~~~~ out 0.50 Ramp 100 TFC Wash 7 2:45 90 1.50 Step 100 ~~~~ out0.50 Ramp 100 Equilibrate

TABLE 5 LC Gradient details for Opiates Loading Eluting Step Start SecFlow Grad % A % B % C % D Tee Loop Flow Grad % A % B % C % D Comment 10:00 25 1.50 Step 100 ~~~~ out 0.70 Step 100 Sample clean-up 2 0:25 50.10 Step 100 ~~~~ out 0.50 Step 100 Lower flow rate 3 0:30 90 0.10 Step100 T in 0.50 Step 100 Transfer 4 2:00 15 1.50 Step 50 50 in 0.50 Ramp84 16 Loop fill/Grad Elute 5 2:15 15 1.50 Step 50 50 in 0.50 Ramp 67 33Loop fill/Grad Elute 6 2:30 15 1.50 Step 100 out 0.50 Ramp 50 50 TFCWash/Grad Elute 7 2:45 15 1.50 Step 100 out 0.50 Ramp 34 66 TFCWash/Grad Elute 8 3:00 15 1.50 Step 100 out 0.50 Ramp 18 82 TFCWash/Grad Elute 9 3:15 15 1.50 Step 100 out 0.50 Ramp ~ 100 TFCWash/Grad Elute 10 3:30 110 1.50 Step 100 out 0.50 Step 100 Equilibrate11 5:20 60 1.50 Step 100 out 0.70 Step 100 Equilibrate

TABLE 6 LC Gradient details for Amphetamines Loading Eluting Step StartSec Flow Grad % A % B % C % D Tee Loop Flow Grad % A % B % C % D Comment1 0:00 25 1.50 Step 100 ~~~~ out 0.70 Step 100 Sample clean-up 2 0:25 50.10 Step 100 ~~~~ out 0.50 Step 100 Lower flow rate 3 0:30 90 0.10 Step100 T in 0.50 Step 100 Transfer 4 2:00 30 1.50 Step 50 50 in 0.50 Ramp84 16 Loop fill/Grad Elute 5 2:30 30 1.50 Step 50 50 in 0.50 Ramp 67 33Loop fill/Grad Elute 6 3:00 30 1.50 Step 100 out 0.50 Ramp 50 50 TFCWash/Grad Elute 7 3:30 30 1.50 Step 100 out 0.50 Ramp 34 66 TFCWash/Grad Elute 8 4:00 30 1.50 Step 100 out 0.50 Ramp 18 82 TFCWash/Grad Elute 9 4:30 30 1.50 Step 100 out 0.50 Ramp ~ 100 TFCWash/Grad Elute 10 5:00 110 1.50 Step 100 out 0.50 Step 100 Equilibrate11 6:50 60 1.50 Step 100 out 0.70 Step 100 Equilibrate

TABLE 7 LC Gradient details for Vitamin D metabolites Loading ElutingStep Start Sec Flow Grad % A % B % C % D Tee Loop Flow Grad % A % B % C% D Comment 1 0:00 30 1.50 Step 80 20 ~~~~ out 0.50 Step 80 20 Sampleclean-up 2 0:30 90 0.10 Step 80 20 T in 0.40 Step 80 20 Transfer 3 2:0060 0.10 Step 100 ~~~~ in 0.50 Ramp 5 95 TFC Wash/Grad Elute 4 3:00 601.50 Step 100 ~~~~ in 0.50 Step 5 95 Loop fill/Grad Elute 5 4:00 1201.20 Step 80 20 ~~~~ out 0.50 Step 80 20 Equilibrate

TABLE 8 MS Conditions for Immunosuppressants Spray voltage 4500Vaporizer Temperature 350 Sheath Gas 60 Aux gas 35 Capillary Temperature200 S-lens amplitude 55

TABLE 9 MS Conditions for Vitamin D Metabolites Spray voltage 5000Vaporizer Temperature 400 Sheath Gas 60 Aux gas 35 Capillary Temperature199

TABLE 10 MS Acquisition Settings for Vitamin D Metabolites (IonMonitoring: SRM) Precursor Ion Product Ions Collision Analyte (Q1) (Q3)Energy S-lens Vitamin D2 413.353 91.098 55 87 95.158 33 87 105.104 42 87159.149 28 87 Vitamin D3 401.352 91.122 50 87 105.133 36 87 159.139 2487 365.425 10 87 d6-Vitamin 407.380 107.115 30 87 D3 133.105 31 87147.199 27 87 159.190 27 87

TABLE 11 Acquisition Method Parameters for Vitamin D Metabolites Scanwidth (m/z) 0.01 Scan time (s) 0.05 Peak width (Q1) 0.2 Peak width (Q3)0.7 Chrom peak filter width 10 Collision Pressure 1.5

Referring to Tables 4-7, it was found that selecting the amount oforganic in the transfer loop of valve one (what it takes to getcompounds off the turboflow column in a reasonable time frame), createdsignificant efficiency in transfer, because the loop was optimallypre-loaded. It was also found that this value is unique to each class ofcompounds.

Likewise, referring to Tables 4-7, it was also found that the ratio ofthe flow rates between the clean-up elution step (i.e., the turbulentflow chromatography elution step) and the analytical column loading step(the whole thing can be called the transfer step) was optimized perindividual compound class to ensure that everything eluting from theclean-up column was focused on the head of the analytical column (i.e.,no analytical elution during the transfer step). This was found tosignificantly improve recovery and analytical peak shape.

Likewise, referring to Tables 4-7, it was also found that the analyticalelution step could be optimized for each compound class to separateisobaric interferences, cross-talk between compounds, and matrixinterferences.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

What is claimed is:
 1. A method, comprising: providing a sample containing two or more analytes, the two or more analytes comprising at least one analyte selected from a first group and at least one analyte selected from a second group, wherein respective log partition coefficient (logP) values of the two or more analytes are separated by at least 4.5 logP units; selecting pre-set system parameters from a control system for chromatographic separation and mass spectrometric analysis of the two or more analytes, the pre-set system parameters being configured for chromatographic separation and mass spectrometric analysis of analytes selected from the first group and the second group spanning a logP range of −1.2 to 6; purifying the two or more analytes based on liquid chromatography system parameters selected from the pre-set system parameters using a single analytical liquid chromatography column of a liquid chromatography system, one aqueous mobile phase buffer solution of the liquid chromatography system, and one organic or non-aqueous mobile phase buffer solution of the liquid chromatography system; and analyzing the two or more analytes using a mass spectrometer based on selected mass spectrometry system parameters.
 2. The method of claim 1, wherein purifying the two or more analytes comprises varying one or more liquid chromatography system parameters particular to each analyte, wherein the liquid chromatography system parameters are selected from the group consisting of mobile phase buffer flow rate, a ratio of the aqueous mobile phase buffer solution to the organic or non-aqueous mobile phase buffer solution, a gradient varying ratios of the aqueous and organic or non-aqueous mobile phase buffer solutions, and combinations thereof.
 3. The method of claim 1, wherein the two or more analytes are separated by 4.5 to about 6 logP units.
 4. The method of claim 1, wherein the two or more analytes are separated by about 6 to about 7 logP units.
 5. The method of claim 1, wherein the two or more analytes are separated by at least 6.84 logP units.
 6. The method of claim 1, wherein the two or more analytes are selected from vitamin D, steroid hormones, protein hormones, proteins, peptide hormones, peptides, bacterial toxins, immunosuppressants, chemotherapeutics, tricyclic antidepressants, azole antifungals, anti-epileptics, anti-retrovirals, opiates and/or opioids, drugs of abuse, barbiturates, benzodiazepines, metabolites thereof, and combinations thereof.
 7. The method of claim 1, wherein the two or more analytes include 25-hydroxy vitamin D₂ and bisulfan.
 8. The method of claim 1, wherein the sample contains a first analyte, a second analyte, and a third analyte, wherein the first analyte has a logP in a range of −1.2 to 0, the second analyte has a logP in a range from 0 to 5, and the third analyte has a logP greater than 5 and less than or equal to
 6. 9. The method of claim 1, wherein the sample comprises a biological fluid sample.
 10. The method of claim 9, wherein the biological fluid sample is selected from the group consisting of blood, plasma, deproteinated plasma, serum, deproteinated serum, sputum, bile, saliva, urine, feces, tears, perspiration, a swab from a body site, or a tissue sample.
 11. The method of claim 1, wherein the control system is configured to control or vary at least one of mobile phase buffer flow rate, a ratio of the aqueous mobile phase buffer solution to the organic or non-aqueous mobile phase buffer solution, a gradient varying ratios of the aqueous and organic or non-aqueous mobile phase buffer solutions, and combinations thereof.
 12. The method of claim 1, wherein the one aqueous mobile phase buffer solution and the one organic or non-aqueous mobile phase buffer solution each include a source of ammonium ions.
 13. The method of claim 12, wherein the source of ammonium ions in the one aqueous mobile phase buffer solution is ammonium formate or ammonium acetate and the source of ammonium ions in the one non-aqueous mobile phase buffer solution is ammonium formate or ammonium acetate.
 14. The method of claim 1, wherein: the one aqueous mobile phase buffer solution comprises: water; a source of ammonium ions; and an acid, and t he one organic or non-aqueous mobile phase buffer solution comprises: an organic phase; a source of ammonium ions; and an acid.
 15. The method of claim 14, wherein the source of ammonium ions in the one aqueous mobile phase buffer solution is ammonium formate or ammonium acetate and the source of ammonium ions in the one non-aqueous mobile phase buffer solution is ammonium formate or ammonium acetate.
 16. The method of claim 14, wherein the acid in the one aqueous mobile phase buffer solution and the acid in the one non-aqueous mobile phase buffer solution is formic acid.
 17. The method of claim 14, wherein the organic phase is selected from the group consisting of methanol, acetonitrile, ethanol, isopropanol, and combinations thereof.
 18. The method of claim 14, wherein the organic or non-aqueous mobile phase buffer solution comprises 10 mM ammonium formate with 0.01% formic acid in methanol.
 19. The method of claim 14, wherein the aqueous mobile phase buffer solution comprises 10 mM ammonium formate with 0.01% formic acid in water.
 20. The method of claim 1, wherein the liquid chromatography system further comprises a single sample clean-up liquid chromatography column in fluid communication with and upstream of the single analytical liquid chromatography column.
 21. The method of claim 20, wherein purifying the two or more analytes comprises: introducing the two or more analytes into the single sample clean-up liquid chromatography column; eluting the two or more analytes from the single sample clean-up liquid chromatography column onto the single analytical liquid chromatography column; and eluting the two or more analytes from the single analytical liquid chromatography column into the mass spectrometer, wherein each of the foregoing is performed with one or both of the one aqueous mobile phase buffer solution and the one organic or non-aqueous mobile phase buffer solution.
 22. The method of claim 21, wherein the two or more analytes are eluted from the single analytical liquid chromatography column with a gradient of the organic or non-aqueous mobile phase buffer solution to the aqueous mobile phase buffer solution.
 23. The method of claim 1, wherein the liquid chromatography system is selected from the group consisting of high-performance liquid chromatography (HPLC), ultra high-performance liquid chromatography (UHPLC), high turbulence liquid chromatography (HTLC), and combinations thereof.
 24. The method of claim 1, wherein the mass spectrometer includes an electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) ion source.
 25. A method, comprising: providing a fluid biological sample containing two or more analytes, the two or more analytes comprising at least one analyte selected from a first group and at least one analyte selected from a second group, wherein respective log partition coefficient (logP) values of the two or more analytes are separated by at least 4.5 logP units, the two or more analytes having respective logP values within the range of about −1.2 to about 6; providing a liquid chromatography-mass spectrometry (LC-MS) system, comprising: a liquid chromatography system, comprising a single analytical liquid chromatography column, a single sample clean-up liquid chromatography column in fluid communication with and upstream of the single analytical liquid chromatography column, one aqueous mobile phase buffer solution, and one organic or non-aqueous mobile phase buffer solution, wherein the one aqueous mobile phase buffer solution and the one organic or non-aqueous mobile phase buffer solution each include a source of ammonium ions, wherein the source of ammonium ions is ammonium formate or ammonium acetate; and a mass spectrometer, purifying the two or more analytes using the single sample clean-up liquid chromatography column, the single analytical liquid chromatography column, the one aqueous mobile phase buffer solution, and the one organic or non-aqueous mobile phase buffer solution by varying at least one liquid chromatography system parameter selected from the group consisting of mobile phase buffer flow rate, a ratio of an aqueous mobile phase buffer solution to a non-aqueous mobile phase buffer solution, a gradient varying ratios of the aqueous and non-aqueous mobile phase buffer solutions, and combinations thereof; and analyzing the two or more analytes using the mass spectrometer by varying at least one mass spectrometry system parameter selected from the group consisting of ionization voltage, desolvation temperature, electrode voltage, collision gas temperature, collision gas pressure, collision energy, and combinations thereof.
 26. The method of claim 25, wherein the liquid chromatography system further comprises a control system configured to control or vary the at least one liquid chromatography system parameter, the method further comprising selecting at least one analysis protocol based on the two or more analytes, wherein selecting the at least one analysis protocol includes varying the at least one liquid chromatography system parameter to purify the two or more analytes without making column or buffer changes, wherein the at least one analysis protocol comprise pre-set system parameters for chromatographic separation and mass spectrometric analysis of the two or more analytes, wherein the pre-set system parameters are configured for chromatographic separation and mass spectrometric analysis of analytes selected from the first group and the second group spanning the logP range of −1.2 to
 6. 27. The method of claim 25, wherein the fluid biological sample contains a first analyte, a second analyte, and a third analyte, wherein the first analyte has a logP in a range of about −1.2 to about 0, the second analyte has a logP in a range from about 0 to about 5, and the third analyte has a logP greater than about 5 and less than or equal to about
 6. 28. A liquid chromatography-mass spectrometry (LC-MS) system, comprising: a liquid chromatography system, the liquid chromatography system comprising a single analytical liquid chromatography column, one aqueous mobile phase buffer solution, and one organic or non-aqueous mobile phase buffer solution; a mass spectrometer; and a control system linked to each of the liquid chromatography system and the mass spectrometer and configured to control or vary at least one LC-MS system parameter, the control system having pre-set system parameters for chromatographic separation and mass spectrometric analysis of analytes spanning a log partition coefficient (logP) range of about −1.2 to about
 6. 29. The system of claim 28, wherein the pre-set system parameters comprise at least one protocol that includes instructions for purifying analytes having logP ranging from about −1.2 to about 6 using the single analytical liquid chromatography column, the single aqueous mobile phase buffer, and the single organic or non-aqueous mobile phase buffer.
 30. The system of claim 28, wherein the one aqueous mobile phase buffer solution and the one organic or non-aqueous mobile phase buffer solution each include a source of ammonium ions.
 31. The system of claim 30, wherein the source of ammonium ions comprises ammonium formate or ammonium acetate.
 32. The system of claim 28, wherein the liquid chromatography system further comprises a single sample clean-up liquid chromatography column in fluid communication with and upstream of the single analytical liquid chromatography column.
 33. The system of claim 28, wherein the at least one LC-MS system parameter is selected from the group consisting of: liquid chromatography system parameters selected from the group consisting of mobile phase buffer flow rate, a ratio of the aqueous mobile phase buffer solution to the organic or non-aqueous mobile phase buffer solution, a gradient varying ratios of the aqueous and organic or non-aqueous mobile phase buffer solutions, and combinations thereof; and mass spectrometry system parameter selected from the group consisting of ionization voltage, desolvation temperature, lens amplitude, electrode voltage, collision gas temperature, collision gas pressure, collision energy, and combinations thereof.
 34. The system of claims 28, wherein the control system includes options for automatically selecting the system parameters for chromatographic separation and mass spectrometric analysis based on categorization of two or more analytes of interest.
 35. The system of claim 28, wherein the liquid chromatography system is selected from the group consisting of high-performance liquid chromatography (HPLC), ultra high-performance liquid chromatography (UHPLC), high turbulence liquid chromatography (HTLC), and combinations thereof.
 36. The system of claim 28, wherein the mass spectrometer is capable of ionizing, fragmenting, and detecting one or more parent ions or product ions specific to each analyte purified and eluted from the liquid chromatography system.
 37. The system of claim 28, wherein the mass spectrometer is selected from the group consisting of single quadrupole, triple quadrupole, ion trap, time of flight (TOF), quadrupole-time of flight (Q-TOF), Fourier transform ion cyclotron resonance (FTICR), electrostatic trap, magnetic sector and combinations thereof.
 38. The system of claim 28, further comprising: a sample handling device configured to manage a plurality of samples; and a sample detection device operably coupled to the sample handling device and/or the control system, wherein the sample detection device is configured to do at least one of: i. identify samples entering the system; ii. identify an analyte in the samples entering the system; iii. select an assay protocol based on the analyte; <iv. direct the sample handling device and/or the control system to initiate analysis of the analyte in the sample; v. direct the control system to select one or more reagents based upon the assay protocol selected for the analyte; vi. direct the control system to select a liquid chromatography mobile phase condition based upon the assay protocol selected for the analyte and cause the liquid chromatography system to purify the analyte; vii. direct the control system to select a mass spectrometer setting based upon the assay protocol selected for the analyte and cause the mass spectrometer to create mass spectral data associated with the selected analyte; or direct the control system to analyze the mass spectral data associated with the selected analyte to identify the presence and/or concentration of the analyte. 